Various types of therapeutic agents based on oligonucleotides such as antisense oligonucleotide (ASO), RNA interference (hereinafter, referred to as ‘RNAi’), microRNA (miRNA), etc., have been actively developed at present.
The ASO has a function of controlling information transfer from a gene into protein by changing an intermediate metabolism of mRNA through single-stranded RNA or DNA strand, which is to achieve desired expression control of target protein by selecting base sequences that are sufficiently complementary and specifically hybridized. The ASO is sequence-specifically bonded to a target gene, which does not have an effect on expression of other genes other than the target gene. Therefore, the ASO technology is a tool that is useful for analysis of functions in vivo of the specific protein, and has a possibility of being utilized as a gene therapy with respect to specific diseases (FASEBJ. 9, 1288-1296, 1995).
miRNA (microRNA) is small RNA of a large group that is naturally produced in a subject, and at least some of them control the expression of the target gene. The miRNA is formed from approximately 70 nucleotide single-stranded hairpin precursor transcriptomes by ribonuclease dicer (Ambros et al. 2003. current biology 13(10):807-818), wherein the dicer cuts the precursor to form 21-23 nucleotide double-stranded miRNAs. In many cases, the miRNA is transcribed from some of DNA sequences of which functions have not been found yet. The miRNA is not translated into protein, but rather, the miRNAs are combined with the specific mRNAs that block the translation. It is though that the miRNAs incorrectly form base pairs with targets thereof to inhibit the translation.
Since a role of the RNAi had been found, it was found that the RNAi sequence-sequentially functions to mRNA in various kinds of mammalian cells (Silence of the transcripts: RNA interference in medicine. J Mol Med, 2005, 83: 764-773). When a long chain of double-stranded RNA is delivered to a cell, the delivered double-stranded RNA is converted into a small interfering RNA (hereinafter, referred to as ‘siRNA’) which is processed to 21 to 23 base pairs (bp) by an endonuclease called a Dicer. siRNA has a short chain of double-stranded RNA having 19 to 27 bases and is coupled to an RNA-induced silencing complex (RISC), whereby a guide (antisense) strand recognizes and decomposes a target mRNA to sequence-specifically inhibit the expression of the target gene (Nucleic-acid therapeutics: basic principles and recent applications. Nature Reviews Drug Discovery. 2002. 1, 503-514).
In particular, the long-chain of double-stranded RNA delivered from the outside has a problem of excessively causing a non-sequence-specific immune reaction through interferon expression in a mammal cell; however, it is found that the problem may be overcome by a short-stranded siRNA (Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001. 411, 494-498).
However, siRNA also has a possibility of innate immune response stimulation generated through sensors present in cells, and accordingly, in order to overcome the possibility, the specific structure of the siRNA is changed or 2-methoxy substituents or 2-fluoro substituents have been developed.
A chemically synthesized siRNA has a double-stranded of about 19 to 27 base pairs (bps) and has a 2-nt(nucleotide) overhang structure at 3′-end, and in order that the double-stranded siRNA expresses an activity, it is known that the double-stranded siRNA has a structure consisting of 3′-hydroxyl group (OH) and 5′-phosphate group (PO4) (Effect of asymmetric terminal structures of short RNA duplexes on the RNA interference activity and strand selection. Nucleic Acids Res 1 Oct. 2008:5812-5821). It is known that a commercialized and synthesized siRNA has a structure in which hydroxyl groups are present at both ends, and when the synthesized siRNA is delivered to a cell, siRNA 5″-end is phosphorylated by a phosphorylation kinase to express functions of siRNA (siRNA function in RNAi: A chemical modification analysis. RNA 2003. 9: 1034-1048).
Bertrand et al., found that as compared to an antisense oligonucleotide (ASO) on the same target gene, siRNA has an effect of significantly inhibiting mRNA expression in vitro and in vivo, and the corresponding effect is maintained for a long time (Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem. Biophys. Res. Commun. 2002. 296: 1000-1004).
In addition, since siRNA is complementarily coupled to a target mRNA to sequence-specifically regulate the expression of the target gene, a mechanism of the siRNA has an advantage that a target to be capable of being applied may be remarkably increased as compared to the existing antibody-based medical product or small molecular drug. (Progress Towards in Vivo Use of siRNAs. MOLECULAR THERAPY. 2006 13(4):664-670).
Even if an oligonucleotide (siRNA, etc)-based therapeutic agent has excellent effect and variously usable range, in order to develop the siRNA as a therapeutic agent, the siRNA is required to be effectively delivered to the target cell by improving stability of siRNA and a cell delivery efficiency of siRNA (Harnessing in vivo siRNA delivery for drug discovery and therapeutic development. Drug Discov Today. 2006 January; 11(1-2):67-73).
In particular, since the oligonucleotides such as siRNA, etc., are not passed through a hydrophobic phospholipid bilayer of a cell due to negative charges thereof, it is difficult to be delivered in the cell through a simple diffusion.
In order to increase a delivery efficiency of the oligonucleotides in vivo or in vitro, various kinds of cell delivery materials have been developed. Liposomes, cationic surfactants, etc., are generally and largely used. Further, methods of using a carrier such as a method of fusing a gene in liposome, a method of using a cationic lipid or a cationic polymer, etc., a method of changing a chemical structure, that is, changing a combined basic structure of oligonucleotide to methylphosphonate, peptide nucleic acid (PNA), etc., and a method of using a conjugate have been known (Chemically modified siRNA: tools and applications. Drug Discov Today. 2008 October; 13(19-20):842-855; Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic Acids Res. 2008 July; 36(12):4158-71).
Among them, a method of using a nanocarrier, that is, a method of using various polymers such as liposome, cationic polymer composite, etc., is to capture oligonucleotides in a nanocarrier by forming nanoparticles to deliver the captured oligonucleotides to cells. Among the methods of using the nanocarrier, a method of using polymeric nanoparticle, polymer micelle, lipoplex, or the like, is mainly used, wherein the lipoplex consists of cationic lipid to interact with anionic lipid of endosome of a cell, thereby inducing a destabilization effect of the endosome to deliver the siRNA into a cell (Mechanism of oligonucleotide release from cationic liposomes. Proc. Natl. Acad. Sci. USA. 1996 Oct. 15; 93(21):11493-8).
In particular, it is known that when the oligonucleotide is siRNA, chemical materials, etc., are connected to end portions of a siRNA passenger (sense) strand to provide increased pharmacokinetics characteristics, such that high efficiency may be induced in vivo (Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004 Nov. 11; 432(7014):173-8). Here, stability of the siRNA may vary depending on properties of the chemical materials bonded to ends of the siRNA sense (passenger) or antisense (guide) strand. For example, an siRNA to which a polymer compound such as polyethylene glycol (PEG) is conjugated, interacts with an anionic phosphate group of siRNA in the presence of cationic materials to form a complex, thereby being a carrier having an improved siRNA stability (Local and systemic delivery of VEGF siRNA using polyelectrolyte complex micelles for effective treatment of cancer. J Control Release. 2008 Jul. 14; 129(2):107-16). In particular, micelle consisting of polymer complexes has an extremely small size, significantly uniform distribution, and is spontaneously form, thereby being easy to manage quality of formulation and secure reproducibility, as compared to other systems used as a drug delivery vehicle, such as microsphere, nanoparticle, etc.
Further, in order to improve an intracellular delivery efficiency of siRNA, technology of using a siRNA conjugate in which hydrophilic material which is a biocompatible polymer (for example, polyethylene glycol (PEG)) is conjugated to the siRNA by a simple covalent bond or a linker-mediated covalent bond, to thereby secure stability of siRNA and have effective cell membrane permeability was developed (see Korean Patent Publication No. 883471). However, the chemical modification of the siRNA and the conjugation with the polyethylene glycol (PEG) (PEGylation) still has disadvantages that stability in a living body is low and delivery into a target tissue is not smooth.
In order to solve the disadvantages, a structure comprising double-stranded oligo RNA (‘double-stranded oligo RNA structure’) in which a hydrophilic material and a hydrophobic material are bonded to oligonucleotides, in particular, double-stranded oligo RNA such as siRNA, was developed, wherein the double-stranded oligo RNA structure forms a self assembled nanoparticle named a self assembled micelle inhibitory RNA (SAMiRNA™) by a hydrophobic interaction of a hydrophobic material (see Korean Patent Publication No. 1224828), the SAMiRNA™ technology has an advantage in that homogenous nanoparticles having a significantly small size are capable of being obtained as compared to the existing delivery technologies.
As a specific example of the SAMiRNA™ technology, PEG (polyethylene glycol) is used as a hydrophilic material, wherein PEG is synthetic polymer, which is used for increasing solubility of pharmaceuticals, particularly, protein, and for controlling pharmacokinetics. PEG is a polydisperse material as like all synthetic polymers, a polymer in one batch consists of the sum of different number of monomers, a molecular weight is shown in the Gaussian curve, and polydispersity index (Mw/Mn) expresses the homogeneity degree of a material. That is, when PEG has a low molecular weight (3 to 5 kDa), the polydisperse index is about 1.01, and when PEG has a high molecular weight (20 kDa), the polydisperse index is about 1.2 which is high, such that as the molecular weight is higher, the homogeneity of the material is relatively low (F. M. Veronese. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials (2001) 22:405-417).
Accordingly, when the PEG is bonded to the pharmaceuticals, polydispersity characteristic of PEG is reflected on the conjugate, such that it is difficult to verify a single material. Recently, in order to overcome the problem, production of materials having a low polydispersity index by synthesizing PEG and improving purification processes is on a rising trend, but has still problems due to polydispersity characteristic of the material, particularly, when PEG is bonded to a material having a small molecular weight, there is difficulty in confirming whether or not the binding is easily performed, etc. (Francesco M. Veronese and Gianfranco Pasut. PEGylation, successful approach to drug delivery. DRUG DISCOVERY TODAY (2005) 10(21):1451-1458).
Further, the size of nanoparticles significantly affects the deliver efficiency into the target cell, when the size of nanoparticles is 50 nm or less, the nanoparticles are rapidly removed from the body via excretion, and when the size of nanoparticles is 100 nm or more, the nanoparticles are not evenly delivered into the tissue, and the effect is reduced. Therefore, it is required to form nanoparticles each having a predetermined size (Wim H De Jong and Paul J A Borm. Int J Nanomedicine. 2008 June; 3(2): 133-149.).
Therefore, the market on a novel concept of oligonucleotide delivery technique for solving polydispersity characteristic of hydrophilic materials such as PEG, etc., of themselves while maintaining the excellent effects of SAMiRNA™ according to the related art as they are, has been urgently demanded.